Significance Previous studies have shown that the nef gene of SIV is important for maintaining high virus load and progression to simian AIDS (SAIDS) in adult rhesus macaques. Our analysis of SIV clones with large deletions in the Nef gene reveals strong selection pressure to restore the Nef translation frame in vivo and to produce disease. This finding raises serious concerns for live-attenuated viral vaccines based on deletion clones of HIV. Objectives Live-attenuated viral vaccines for simian immunodeficiency virus (SIV) have been constructed by deleting viral accessory genes, including nef, that are important for pathogenesis in rhesus macaques. In previous studies, such deletion viruses, as well as replication-competent vectors expressing the cytokines gamma-interferon or interleukin-2 (IL-2), appeared to be relatively safe in juvenile and adult macaques. Results Our study demonstrated that macaques infected with a virus with a large deletion in nef (SIVmac239D153nef) or with SIV vectors, in which a portion of the nef gene was replaced with the gene for IL-2, can develop high virus loads and progress to fatal simian AIDS (SAIDS). Viruses recovered from several animals with disease produced a novel truncated form of Nef protein, designated tNef. Sequence analysis revealed changes in SIVmac239D153nef and the viral vectors expressing IL-2 that restored the translation frame to produce tNef. Juvenile macaques infected with serially passaged virus expressing tNef exhibited high virus load and signs of immunodeficiency; however, macaques infected with a molecular clone constructed by replacing nef of SIVmac239 with tNef sequences showed low virus load. Thus, expression of tNef may be necessary but not sufficient for high virus load and pathogenesis. Taken together, these findings demonstrate strong selective pressure to restore pathoge nic potential in live-attenuated primate lentivirus vaccines. Future Directions Changes in the viral genome in pathogenic viruses lacking full Nef will be defined. Such changes, involving either another viral gene or a viral regulatory element, may compensate for loss of full Nef function in vivo. In vitro tissue culture studies will be done to develop novel assays for Nef function. In vivo assessments will involve inoculating rhesus macaques with SIV clones containing compensating changes for Nef. KEY WORDS nef gene, simian AIDS, SIV clones FUNDING NIH Grant AI38532